Use of neurotoxin therapy for treatment of urologic and related disorders

ABSTRACT

The present invention relates to methods for treating neurological-urological conditions. This is accomplished by administration of at least one neurotoxin.

This application is a continuation of U.S. patent application Ser. No.09/463,040, filed Jan. 17, 2000 now U.S. Pat. No. 6,365,164, which is a371 of PCT Application No. PCT/US98/14625, filed Jul. 15, 1998, whichclaims priority under 35 U.S.C. § 119(e) to U.S. Provisional ApplicationSerial No. 60/052,580, filed Jul. 15, 1997. The entire disclosure ofU.S. patent application Ser. No. 09/463,040 is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention provides methods for treating neuronally-mediatedurologic and related disorders, for example, benign prostatichyperplasia (BPH) and prostatitis. This is accomplished by administeringa composition that includes at least one neurotoxic compound or byconventional therapies.

BACKGROUND OF INVENTION

Many medical conditions in urology are rooted in a spastic dysfunctionof the sacral reflex arcs. Examples of such conditions include pelvicpain (e.g., interstitial cystitis, endometriosis, prostatodynia,urethral instability syndromes), pelvic myofiscial elements (e.g.,levator sphincter, dysmenorrhea, anal fistula, hemorrhoid), urinaryincontinence (e.g., unstable bladder, unstable sphincter), prostatedisorders (e.g., BPK prostatitis, prostate cancer), recurrent infection(secondary to sphincter spasticity), and urinary retention (secondary tospastic sphincter, hypertrophied bladder neck) and neurogenic bladderdysfunction (e.g., Parkinson's Disease, spinal cord injury, stroke,multiple sclerosis, spasm reflex).

The prostate is a partially glandular and partially fibromuscular glandof the male reproductive system. During aging, the prostate tends toenlarge (hypertrophy). This prostatic enlargement can lead to urethralobstruction and voiding dysfunction.

Prostatic enlargement is a common occurrence in older men. Lytton et al(Lytton, B., Emery, J. M. and Harvard, B. M. [1973] 99: 639-645)estimated that a 45 year old male had a 10% risk of prostate surgery byage 70. The U.S. Census Report estimates that there are 30 millionpeople today over age 65. This segment of the population is projected torise to 50 million over the next 30 years. Therefore, the number of menwith prostatic enlargement also will increase. According to draftreports of the National Kidney and Urologic Disease Advisory Board,425,000 prostatectomies were performed in the United States in 1989.Based on population growth estimates, the number of prostatectomiesperformed annually will rise to 800,000/year by the year 2020.

The urethra passes through the prostate (prostatic urethra) as itcourses to the external urethral orifice. The prostate has five distinctlobes that are apparent at 12 weeks in the human fetus (Lowsley, O. S.Am. J. Anat. [1912] 13: 299-349.). Although the lobular branching foundin the fetus is not visible in the prepubescent prostate, the lateralmiddle anterior and posterior lobes are used to describe the enlargedprostate.

A more recent viewpoint is that the prostate also is comprised ofseveral morphologically distinct zones (McNeal, J. Urol. Clin. North Am.[1990] 17(3): 477-486). The majority of the glandular volume is composedof the peripheral zone (˜70-75%). The remainder of glandular volume isdivided into the central zone (˜20-25%), the transition zone (˜5-10%)and the periurethral glandular zone (˜1%).

McNeal (1990) reported that BPH develops in the transition zone and theperiurethral glandular zone. BPH nodules develop either within orimmediately adjacent to the pre-prostatic sphincteric zone. Thetransition zone is a small region close to the urethra intimatelyrelated to the proximal urethral sphincter. The stroma of the transitionzone is dense and compact, and is unusually susceptible toneurologicaly-induced disturbances of growth control. Its glandspenetrate the sphincter, while sphincter muscle fibers penetrate thetransition stroma. The periurethral glandular zone has a similarurogenic sinus origin as the transition zone.

BPH may be associated with increased amounts of stroma relative toepithelium (Bartsch, G., Muller, H. R., Oberholzer, M., Rohr, H., P., J.Urol. [1979] 122: 487-491). A significant portion of the stroma issmooth muscle (McNeal, 1990) which is under sympathetic nervous control.The contractile properties of this smooth muscle could account for thedynamic component of obstruction in BPH (Bruschini, H. et al. [1978]Invest. Urol. 15(4): 288-90; Lepor, H. [1990] Urol. Clin. North Am.17(3): 651-658).

In addition to sympathetic control of prostatic stroma, the prostate ishighly innervated. The prostate nerve fibers enter the prostate from theposterior lateral aspect, with a concentration of ganglia near thejunction between the prostate and the seminal vesicles (Maggi, C. A.,ed. [1993] Nervous control of the Urogenital System, Harwood AcademicPublishers; Higgins, J. R. A. and Gosling, J. A. [1989] Prostate Suppl.2: 5-16).

Acetylcholine (ACH), neuropeptide Y (NPY), vasoactive intestinal peptide(VIP) and noradrenaline fibers have been described in this gland. A richplexus of ACH-positive nerve cell bodies is associated with secretoryacini in all parts of the gland. Some of the ACH fibers also contain NPYneurons. VIP-containing neurons have been found associated withACH-containing nerve cell bodies. Occasional neurons have been foundbetween the ACH-staining nerve fibers, suggesting that both NPY andnoradrenergic neurons supply smooth muscle (Higgins, J. R. A. andGosling, J. A. [1989] Prostate Suppl. 2: 5-16).

Autonomic nerves are distributed evenly between the central andperipheral zones of the prostate (Higgins, J. R. A. and Gosling, J. A.[1989] Prostate Suppl. 2: 5-16). Peripheral neuronal control is similar.In addition, there is no difference in the type of nerve fibers foundassociated with either epithelial or stromal elements of the gland.

The anatomical studies of nerve fiber types in the prostate, coupledwith other studies of innervation of prostatic stroma (Brushing H.,Schmidt, R. A., Tanagho, E. A., [1978] Invest. Urol. 15(4): 288-290;Watanabe, H., Shima, M., Kojima, M., Ohe, H. L. [1989] Pharmacol. Res.21(Suppl 2): 85-94) suggest that cholinergic innervation influencesepithelial behavior, while adrenergic innervation influences stromaltonus (excitability). These observations have provided a rationale forthe use of for example, alpha blockers in the treatment of BPIL Theeffects of alpha blockers (Downie, J. W. and Bialik, G. J. [1988] J.Pharmacol. Exp. Ther. 246(1): 352-358) can also account for improvementsin symptoms of BPH as a result of dampening of dysfunctional striatedsphincter behavior by the alpha blockers.

Studies have also shown that there are several tachykinins (for example,substance P [SP], calcitonin gene related peptide [CGRP], neurokinin A,bradykinin, and nerve growth factor [NGF]) that can influence the tonusof smooth muscle (Hakanson, et al.,[1987] Neuroscience 21(3): 943-950).Neurotransmitter receptors have been quantified throughout the prostate(e.g., NPY, VIP, SP, leu-enkephalin (L-enk), met-enkephalin, 5-HT,somatostatin, acetylcholinesterase positive fibers (ACTH), and dopaminebeta-hydroxylase (DBH) (Crowe, R., Chapple, C. R., Burnstock, G. TheHuman Prostate Gland: A Histochemical and Immunohistochemical Study ofNeuropeptides, Serotonins, Dopamine beta-Hydroxylase andAcetylcholinesterase in Autonomic Nerves and Ganglia). There is somevariation in receptor density at different prostatic sites in benignprostatic hyperplasia.

Changes in electrophysiologically recorded cellular behavior and inconcentration of neuropeptides within the spinal cord have been shown tobe a secondary consequence of mechanical pinch to the tail muscles of arat, catheter stimulation of the posterior urethra, andelectrostimulation of a peripheral nerve. Dyssynergia between thedetrusor and the urethral sphincter is a significant finding inprostatodynia patients. Denervation of the prostate has been shown toproduce dramatic changes within the prostatic epithelium. Thus there isevidence that experimentally induced alterations in neurologicalinfluences can be produced in the sacral, spinal cord, bladder orurethra through mechano-, electro-, chemical or thermal (microwave,laser) methods to change irritative behavior. However, there have beenno known attempts to use neurotoxins for therapeutic applications.

There is poor correlation between the degree of prostatic enlargementand the severity of symptoms. While 80% of men age 70 show BPH ontransrectal ultrasound scans, only 20% seek surgery (Coffey, D. S. andWalsh, P. C. [1990] Urol. Clin. North Am. 17(3): 461-475), the treatmentof choice for BPH (Fowler, F. J. Jr., Wennberg, J. E., Timothy, R. P.[1988] J. Amer. Med. Assoc., 259(20): 3022-3028). Symptoms of irritationmay far exceed symptoms expected based on the size of the prostate.Symptoms may improve after surgical treatment of BPH by procedures suchas transurethral resection of the prostate (TURP) (Christensen, Aagaard,M. M. J., Madsen, P. O. [1990] Urol. Clip North Am. 17(3): 621-629),balloon dilation (Dowd, J. B. and Smith, J. J. III [1990] Urol. Clin.North Am. 17(3): 671-677), or prostatic hyperthermia (Baert, L., Ameye,F., Willemen, P., et al., [1990] J. Urol. 144: 1383-1386). However,symptoms persist in as many as 15% of all BPH patients (Baert, L.,Ameye, F., Willemen, P., et al., [1990] J. Urol. 144: 1383-1386;Wennberg, J. E., Mullly, A. G., Hanley, D., Timothy, R. P., Fowler, F.J., Roos, R. P., Barry, M. J. et al., [1988] J. Amer. Med. Assoc. 259:3027-3030). Up to 25% of BPH patients have secondary procedures in longterm follow-up studies, suggesting that surgical approaches do notaddress the fundamental mechanisms that produce BPH, i.e., the faultyneurological influence (control mechanism) on the integrity of the lowerurinary tract.

The need for repeated surgeries, the morbidity and mortality associatedwith TURP and the cost of surgery have led to the development of somenon-surgical approaches such as androgen ablation (McConnell, J. D.,[1990] Urol. Clin North Am. 17(3): 661-670) and the use of alphablockers discussed above, but few medical or surgical treatments to datehave produced a restoration of void behavior to normal state (flow rateof about 25 cc/sec and void volume of about 400 cc).

The present invention uses chemical and non-chemical methods,particularly neurotoxins, to modulate neuronally-mediated urologic andrelated disorders. For example, such methods can be used to treat BPHand related conditions such as prostatitis. The instant invention alsomay remove triggers of changes in the CNS by non-chemical methodsincluding biofeedback, or by chemical methods that treat BPH and otherurological conditions by the administration of substances that blockvarious neurological activities, such as, for example, selectedneurotoxins.

OBJECTS AND SUMMARY OF THE PRESENT INVENTION

It is an object of the instant invention to provide safe, inexpensive,out patient methods for the prevention and treatment ofurological-neurological dysfunctional states or conditions, for example,prostatic enlargement.

It is a farther object of the present invention to provide compositionsfor this therapeutic goal. It is a still further object of the presentinvention to provide dosages and methods of administration forcompositions useful for the prevention and treatment ofneurological-urological conditions.

Other objects of the present invention will be readily apparent to thoseof ordinary skill in the art.

In accordance with one aspect of the present invention, there areprovided methods of treating urological-neurological conditions inmammals, said methods comprising the step of administering atherapeutically effective amount of at least one neurotoxin to such amammal. It is preferred that the neurotoxin inhibits synaptic function.Such inhibition produces selective denervation, and, for example,atrophy of the prostate and reversal of irritative symptoms associatedwith prostatic enlargement. In one embodiment of the instant invention,the neurotoxin induces dysfunction of the presynaptic neuronal terminalby specific binding and blockade of acetylcholine release at myoneuraljunctions. Such a neurotoxin can be, for example, botulinum toxin type A(e.g., BOTOX, Allergan).

Preferably, the neurotoxin is safe, highly selective and easy todeliver, including when combined with other therapies. Other usefulneurotoxins include capsaicin, resinoferatoxin and α-bungotoxin.Delivery of the neurotoxin can be by any suitable means. A convenientand localized method of delivery is by injection.

A therapeutically effective amount of the neurotoxin is the dosagesufficient to inhibit neuronal activity for at least one week, morepreferably one month, most preferably for approximately 6 to 8 months orlonger. Dosing can be single dosage or cumulative (serial dosing), andcan be readily determined by one skilled in the art. Neurotoxin can bedelivered serially (i.e., one time per month, one time per every sixmonths) so that the therapeutic effect can be optimized. Such a dosageschedule is readily determined by one skilled in the art based on, e.g.,patient size and the condition to be treated, and will depend on manyfactors, including the neurotoxin selected, the condition to be treated,the degree of irritation, and other variables. One suggested course oftreatment for BPH is 200 units every three days up to the LD50 for Botoxor about 2500 units.

The aforementioned methods of treatment should be particularly usefulfor the long-term control of neurological-urological disorders, e.g.,the symptoms of prostatic enlargement, without the need for surgicalintervention. Furthermore, the methods of the instant invention providefor control of neurological-urological disorders, e.g., BPH and relatedconditions, in a highly selective manner, without the potential sideeffects and treatment failures associated with current treatmentmodalities.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

“Urological-neurological condition or disorder” includes many medicalconditions in urology rooted in a spastic dysfunction and/ordegeneration of the sacral reflex arcs. Examples of such conditionsinclude pelvic pain (e.g., interstitial cystitis, endometriosis,prostatodynia, urethral instability syndromes), pelvic myofascialelements (e.g., levator sphincter, dysmenorrhea, anal fistula,hemorrhoid), urinary incontinence (e.g., motor or sensory, unstablebladder, unstable sphincter), prostate disorders (e.g., BPH, prostatecancer), recurrent infection (secondary to sphincter spasticity), andurinary retention (secondary to spastic sphincter, hypertrophied bladderneck), and neurogenic bladder dysfunction (e.g., Parkinson's Disease,spinal cord injury, stroke, multiple sclerosis, spasm reflex) and othersuch urological conditions of a nervous etiology.

The prostatic enlargement that can be treated according to the methodsof the instant invention can be of any etiology. The instant inventionis particularly suited for the treatment of prostatic hyperplasia,especially benign prostatic hyperplasia. The present invention can alsobe used for the treatment of enlargement of the prostate withinflammation (prostatitis), particularly abacterial prostatitis. Inaddition, the methods of the instant invention can be used for thetreatment of prostatodynia.

Without being bound by theory, the basis for the treatment of theneurological-urological conditions according to the instant invention isthe removal or modulation of the neural basis for the dysfunctionalregulation of the affected tissue. For example, the modulation of theneural basis of prostate glandular dysfunction can be accomplished byany non-surgical means known in the art. Such means can include, forexample, biofeedback, α-blockers, pharmacological methods, and the useof one or more neurotoxins to inhibit synaptic function in the affectedgland. It is preferred that the neurotoxin cause long-lasting inhibitionof synaptic function, preferably greater than one week, more preferablygreater than one month, most preferably six to eight months or longer.Such neurotoxins can include, for example, capsaicin, resinoferatoxin,α-bungotoxin, terodotoxin and botulinum toxin. Botulinum toxin is apreferred neurotoxin according to the instant invention, particularlybotulinum toxin A, more particularly BOTOX (Allergan).

The toxin can be formulated in any pharmaceutically acceptableformulation in any pharmaceutically acceptable form Such forms andformulations include liquids, powders, creams, emulsions, pills,troches, suppositories, suspensions, solutions, and the like. The toxincan also be used in any pharmaceutically acceptable form supplied by anymanufacturer.

In a preferred embodiment in accordance with the method of the instantinvention, the neurotoxin is botulinum toxin type A Therapeuticallyeffective amounts of botulinum toxin can be any amounts or doses thatare less than a toxic dose, for example, less than about 3000 IU/70 kgmale, preferably between 100 IU/70 kg male to 1200 IU/70 kg. The dosagescan be given as a single dose, or as divided doses, for example, dividedover the course of four weeks.

The neurotoxins of the instant invention can be administered by anysuitable means. In the preferred embodiment of the invention, botulinumtoxin is administered by injection. Such injection can be administeredto any affected area. For example, the neurotoxin can be injectedurethroscopically into the prostate with 200 IU with single or serialdosing. Preferably, the neurotoxin is injected every three days until atherapeutic effect is achieved or up to about 2500 units.

The following techniques are used in this invention:

Tissue Preparation for Light Microscopy

Tissues are fixed in 6% paraformaldehyde in 0.1 M phosphate buffer, pH7.2, for 24 hours, dehydrated in graded alcohol and xylene, and embeddedin paraffin Sections are cut and stained with appropriate stains, suchas hematoxylin/eosin

Tissue Preparation for Electron Microscopy

Tissues are collected and fixed in 2.5% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.2, for 1 hour at 4° C., then incubated with 0.1%osmium tetroxide for 1 hour and embedded in EPON. Ultrathin sections (80nm) are prepared and stained with lead citrate/uranyl acetate andexamined with an electron microscope Philips, model 201).

Tunel Stain for Apoptosis

The tissue is fixed and embedded as described above. The tissues aredeparaffinized and reacted with Proteinase K (Boehringer). They arefurther treated with peroxidase and TDT enzyme and placed in ahumidifier set at 37° C. for one hour. The sections are washed andanti-digoxigenin-peroxidase is added for 30 minutes, followed bystaining with nickel-DAB (diaminobenzene).

Immunohistochemistry Studies

The presence of the neuropeptides VIP, SP, NPY, L-Enk and calcitoningene-related peptide (CGRP) as well as the expression of transforminggrowth factor beta (TGF-beta), transforming growth factor alpha(TGF-alpha), epidermal growth factor (EGF) and basic fibroblast growthfactor (bFGF) are determined in prostatic tissues using appropriatemonoclonal antibodies. Use of neurotoxins results in prostatic atrophy,which should be reflected by lower levels of growth factors in treatedprostatic tissue.

Sections are incubated overnight at room temperature with primaryantibodies followed by immunostaining with avidin-biotin-peroxidase(Veaastain Elite ABC, Vector Labs, USA). Rabbit polyclonal antiserumagainst the neurotransmitters VIP, CGRP, SP, NPY and L-Enk (PeninsulaLabs, USA) is used in these preparations, at dilutions of 1:8000 to1:12,000. Immunocytochemical controls consist of preabsorbing theprimary antiserum with appropriate antigen, or their substitution withnormal serum (Blasi, J., Chapman, E. R., Yamaskai, S., Binz, T.,Niemann, H. and Jahn, R. [1993] The EMBO Journal 12: 4821-4828; Black,J. D. and Dolly, J. O. [1986] J. Cell Biol. 103; 535-544; Linial M.[1995] Is. J. Med. Sci. 31: 591-595). After mounting on slides, sectionsare counterstained with eosin, dehydrated and coverslipped.

Western Blot Analysis of Growth Factor Expression

Treated and untreated prostate cell homogenates are examined forexpression of growth factors by Western blot analysis. Cell homogenateprotein is separated by electrophoresis on SDS-PAGE (7%), thentransferred electrophoretically overnight to nitrocellulose paper(Towbin, H., et al., [1979] Proc. Nat. Acad. Sci. 76(9): 4350-4379). Thenitrocellulose paper is soaked for one hour at room temperature in 0.5%non-fat dry milk dissolved in phosphate buffered saline, and furthersoaked overnight at 4° C. in blocking solution (2% bovine serum albuminin 10 mM Tris/0.15 M NaCl 0.1% sodium azide, pH 7.4). The nitrocellulosemembranes are incubated with antibodies (IgG fractions of anti-TGF-beta,anti-TGF-alpha, anti-EGF and anti-bFGF) purified by protein A (1×10⁶cpm/ml) in blocking buffer for 1 hour. The membrane is washed with PBScontaining Nonidet P40 between incubations. X-O-mat AR2 film (Kodak) isexposed to the membrane at −70° C. and films are developed to examinethe expression of growth factors.

Determination of c-fos and c-myc Expression

Expression of c-fos and c-myc in treated and untreated prostatic tissueis determined by Northern blot analysis as follows. Tissue ishomogenized in lysis buffer for 15 seconds or until the tissuehomogenizes. Sodium acetate is added and the solution is mixed byswirling. An equal volume of water-saturated phenol is added and mixedby inversion, followed by addition of chloroform/isoamyl alcohol. Thesolution is vortexed vigorously for 30 seconds, and allowed to settle onice for 15 minutes. The solution is centrifuged for 10-20 minutes at 4°C. After centrifugation, the aqueous phase is carefully aspirated andplaced in a new polypropylene tube. One volume of isopropanol is addedand the solution is mixed by swirling. The solution is placed in a −20°C. freezer for at least 60 minutes to precipitate RNA. Afterprecipitation, the tube is centrifuged for 10 minutes, and thesupernatant is decanted, leaving the RNA pellet. One ml of ethanol isadded, and the tube is centrifuged for an additional 10 minutes. Theaqueous phase is discarded, and the pellet is washed with 100% ethanolby vortexing. The RNA pellet is redissolved in 0.4 ml of lysis buffer.The RNA is reprecipitated by the addition of 100% ethanol and incubationat −20° C. freezer for at least 60 minutes. The solution is centrifugedand the supernatant discarded. RNA concentration is determined bydiluting 5 μL of sample into 995 μL of DEPC water and measuring theratio of absorbance at 260/280 nm.

The following examples are provided by way of describing specificembodiments without intending to limit the scope of the invention in anyway.

EXAMPLE 1 Denervation of the Prostate

Unilateral denervation of the prostate is carried out by removal of thepelvic ganglia, which overlie the prostate of the rat. This approachpreserves the functional integrity of the bladder and posterior urethraand removes the possibility for artifact arising from major disturbancesin blood flow or micturation. Control animals undergo sham operationswithout concurrent denervation of the prostate. After denervation, theanimals are allowed to recover and maintained prior to collection of theprostate. The prostate is preserved, prepared for light microscopy andexamined histologically. The major findings are (1) reduced epithelialcell height primarily due to a decrease in the clear supranuclear zone(due to a reduction in the amount and size of the apical cisternae andthe endoplasmic reticulum); (2) major changes in protein expression onSDS gel electrophoresis (the endoplasmic reticulum is important inprotein synthesis) (3) modest reduction in the number of secretorygranules; (4) an increase in intracellular vacuoles, intercellular emptyspaces and reduction in microvilli on the cell surface; and (5) asignificant increase in nerve growth factor (NGF) content ipsilateral tothe denervation relative to the control group (188±10 vs. 46±20 vs.29±16 pg/g wet tissue (±SD) NGF is known to influence only sympatheticand sensory neurons. N=15 in both the control and experimental groups.

EXAMPLE 2 Effect of Neurotoxin Injection on Normal Prostate: RatProstate

Rats were randomly assigned into three groups. The first group receiveda single acute dose of Botulinum toxin type A (BOTOX, Allergan) of 5, 10or 15 IU. These animals were sacrificed one week after injection. Thesecond group received a series of 4 weekly injections of 5 IU ofBotulinum toxin and were sacrificed at 5 weeks. Control rats receivedsaline injections. Injections were performed as a single or serialinjections into the left and/or right ventral lobe of the prostate. notethat an injection of methylene blue into one lobe of the rat prostateshowed immediate diffusion into the opposite lobe. Thus, there wascommunication between the prostate lobes and therefore the contralaterallobe could not be used as a true comparative control.

The weight of each prostate ventral lobe collected from healthy animalswas approximately 0.50 gram. All toxin-treated animas showed shrinkageof prostate volume, first in the injected lobe, and with subsequentinjections, reduction in the overall volume. After four serialinjections, the left prostate lobe weighed 0.12-0.17 gram, while theright lobe weighed 0.10-0.14 grams. This represented a reduction of overtwo-thirds of the original size.

EXAMPLE 3 Effect of Neurotoxin Injection on Urological Dysfunctions:Human Data

Three patients with recalcitrant voiding dysfunction were treated withinjections of botulinum toxin (Botox) as follows. Patient 1 was a47-year-old male who was incontinent secondary to an injury sustained atthe cervical vertebrae (level C6-C7) sustained 14 months previously.Urodynamics on presentation revealed a bladder capacity of 30 cc and aweak sphincter (peak urethral pressure of 40 cm water). He had failedmultiple pharmacological regimes and was intolerant to penileclamp/condom devices.

He received four weekly 200 IU botulinum toxin injections into thebladder neck for total dose of 800 IU. Post-injection, his bladdercapacities ranged from 300-400 cc with oxybutinin and 150-200 cc withoutoxybutinin. Peak bladder pressures pre-injection had been 200-cm water,compared to post injection bladder pressures of 40 cm of water. Thepatient was continent with a penile clamp after treatment with botulinumtoxin. In addition, waling and erections improved due to reduced bladderspasticity.

Patient 2 was at 55 year old T12 paraparetic female secondary totraumatic injury 14 years previous. The patient presented with urgeincontinence, and had been on self-catheterization every 2 hours duringthe day and two times at night. The patient received injections into thelateral bladder wall in two weekly injections of 200 IU each for a totalof 400 IU of botulinum toxin. The patient's voiding diary data revealedpre-injection capacities of between 150-200 cc. Post injection, diarydata indicated bladder capacity increased to 300-400 cc. In addition,the patient no longer had annoying constant urge type dysfunction, sleptthrough the night and was continent on self-catheterization every 4hours.

Patient 3 was a 65 year old male with disabling perineal pain followingradiation treatment for prostatic cancer. The patient had failed medicaltherapy. He was treated with one 200 IU injection of botulinum toxininto the external urethral sphincter. The patient experienced a dramaticrelief of testicle pain and had far less severe pain in the shaft of thepenis. Erections were not affected.

EXAMPLE 4 Determination of Smallest Effective Dose

Rats are injected in the prostate ventral lobes with single and serialdoses of botulinum toxin (BOTOX, Allergan). The prostates are harvestedat different time intervals to determine the smallest effective dose, aswell as the morphological and physiological changes taking place withtime. The smallest effective dose is defined as that dose that woulddemonstrate a decrease in prostate volume.

To assess the response to electrical field stimulation, preparations aremounted between two platinum electrodes placed in the organ bath Thetension of the preparations is adjusted. Transmural stimulation ofnerves is performed using a Danted Neuromatic 2000 Stimulator deliveringsingle square-wave pulses at supramaximal voltage with a duration of 0.8milliseconds at a frequency of 0.5 to 80 hertz. The polarity of theelectrodes is changed after each pulse by means of a polarity-changingunit. The train duration is five seconds and the train interval 120seconds. Isometric tension is recorded by using a Gould thermo-array8-channel recorder. Separate experiments are performed to determine thepreload tension producing optimal responses. In addition, the effect ofthe electric field stimulation in the presence of differentconcentrations of individual neuropeptides is determined. Theseneuropeptides are 10-20 μM adrenaline, 10 μM clonidine, 5-50 mMregitine, 10 nM-0.1 μM acetylcholine, 1-3 μM atropine, 1 nM-10 μMnifedipine, 1-10 nM VIP and 1-250 nM NPY. The effect of nitroprusside (anitric oxide releasing substance) and methylene blue (a guanylatecyclase inhibitor) on prostate tone and contraction resulting from fieldstimulation also is examined in these tissues.

EXAMPLE 5 Effect of Botulinum Toxin on Rat Prostatic Tissue: Comparisonof Hormonally Intact Rats to Hormonally Deprived Rats

To determine if there is any interaction between the neurotoxin andtesticularly-derived hormones, studies are performed which will examinethe interaction of the neurotoxin with hormonal components. Thesestudies will compare prostatic tissue treated with botulinum toxinharvested from rats that have undergone orchiectomy (hormonally depletedrate) and prostatic tissue from rats treated with botulinum toxin thatdid not undergo orchiectomy. Fifty-two age-matched rats are treated asdescribed below. Four healthy rats will undergo a sham operationconsisting of anesthesia induction, exposure of the prostate andinjection of 0.2 cc saline into the left ventral lobe of the prostate.Three rats are given bilateral orchiectomy with no injection to theprostate (hormonally depleted controls), five rats will have orchiectomyand injection of 0.2 ml saline in the left ventral lobe (hormonaldepletion+surgical stress control). Four groups of rats receivebotulinum injections of 0.5 IU, 1.0 IU, 1.5 IU and 2.5 IU only(hormonally intact experimental rats). Sixteen rats undergo bilateralorchiectomy. Eight of these rats are treated with a single injection of2.5 IU botulinum toxin into the left ventral lobe 5 weeks after surgery.All rats are sacrificed after six weeks, and the harvested prostate isprepared for examination as described above. A similar atrophic effecton glandular epithelium is expected.

EXAMPLE 6 Effects of Botulinum Toxin on Patients

Patients affected by benign prostatic hyperplasia, abacterial prostatis,or prostatodynia are studied both before and after treatment withbotulinum toxin. Patients are eligible for inclusion in this study ifthey are affected by BPH between the ages of 40 and 80, or if they arebetween 25 and 60 and have been diagnosed with abacterial prostatitis orprostatodynia Preferred patients are those who are not good surgicalcandidates. Patients are evaluated prior to treatment by determinationof prostate specific antigen levels (PSA), evaluation of urodynamicparameters (cystometrogram, urethral pressure profile and flowmetry),determination of American Urological Association (AUA) symptom score(Barry, M. J., et al., [1992] J. Urol., 148: 1549-1557), maintenance ofa voiding diary, and examination of the prostate by transrectalultrasound with biopsy (for BPH patients only). One week after initialevaluation is completed, the patient is injected urethroscopically with200 IU of botulinum toxin as either single unilateral injections, serialunilateral injections or bilateral injections. BPH patients are treatedby TURP or undergo control TURP-biopsy 7 days after single injection or5 weeks after serial injections. The harvested prostatic tissues areprepared for examination as described in Examples 1, 2, 3, and 7-10. Thepatients are reevaluated after injection using the same parametersexamined during the initial evaluation.

The foregoing description of the invention is exemplary for purposes ofillustration and explanation. It will be apparent to those skilled inthe art that changes and modifications are possible without departingfrom the spirit and scope of the invention. All documents cited hereinare hereby incorporated by reference. It is intended that the followingclaims be interpreted to embrace all such changes and modifications.

What is claimed is:
 1. A method of treating urinary incontinence inmammals comprising the step of administering to a mammal that has atleast one symptom of urinary incontinence a therapeutically effectiveamount of a botulinum toxin, thereby treating at least one symptom ofurinary incontinence in said mammal.
 2. The method according to claim 1,wherein the mammal is a human male.
 3. The method of claim 1, whereinthe urinary incontinence is selected form the group consisting ofunstable bladder and unstable sphincter.
 4. The method of claim 1,wherein the botulinum toxin is botulinum toxin type A.
 5. A method fortreating a urinary incontinence disorder, the method comprising the stepof local administration of a botulinum toxin into the urinary tract of apatient that has at least one symptom of urinary incontinence, therebytreating a symptom of urinary incontinence in the patient.
 6. The methodof claim 5, wherein the botulinum toxin is botulinum toxin A.
 7. Themethod of claim 1, wherein the botulinum toxin is administered to thebladder of the mammal.
 8. The method of claim 5, wherein the botulinumtoxin is administered to the bladder of the patient.